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In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonia! transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced [F.sub.1] offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with [F.sub.1] offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation- ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the [F.sub.2] generation. Furthermore, although these [F.sub.1] offspring appeared normal, [F.sub.2] offspring produced by IVF using [F.sub.1] sperm and wild- type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission., Introduction Since its first report in 1978, in vitro fertilization (IVF) has been used in assisted reproductive techniques (ARTs) in humans (1). IVF is often used when a woman's fallopian [...]

Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation (1,2). The whole process, therefore, has never been reproduced in vitro in mammals (3-5), nor in any other species with a very few exceptions in some particular types of fish (6,7). Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas-liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis., Studies on in vitro spermatogenesis date back to organ culture experiments about a century ago (8). In 1937, it was reported that spermatogenesis proceeded up to the pachytene stage of [...]

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods. 10.1126/science.1194174

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.09.041 Byline: Kazuo Yamagata (a), Taiga Yamazaki (a), Hiromi Miki (b), Narumi Ogonuki (b), Kimiko Inoue (a)(b), Atsuo Ogura (a)(b), Tadashi Baba (a) Keywords: Mouse; DNA methylation; Germ cell lineage; Somatic cell cloning; Centric/pericentric region; Epigenetics Abstract: Germ cells have unique features strikingly distinguishable from somatic cells. The functional divergence between these two cell lineages has been postulated to result from epigenetic mechanisms. Here we show that the chromosomal centric and pericentric (C/P) regions in male and female germline cells are specifically DNA-hypomethylated, despite the hypermethylation status in somatic cells. In multipotent germline stem cells, the C/P region was initially hypomethylated and then shifted to the hypermethylation status during differentiation into somatic lineage in vitro. Moreover, the somatic-type hypermethylation pattern was maintained in the somatic cell-derived nuclear transfer embryos throughout preimplantation development. These results imply that the identity of germ cell lineage may be warranted by the hypomethylation status of the C/P region as an epigenetic signature. Author Affiliation: (a) Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan (b) RIKEN, Bioresource Center, Tsukuba Science City, Ibaraki 305-0074, Japan Article History: Received 14 June 2007; Revised 20 September 2007; Accepted 21 September 2007

Germline cells that can form different embryonic stem (ES) as well as embryonic germ (EG) cells are obtained only from embryonic tissues, hence tests were conducted to obtain such cells from other sources. Researchers conducted tests to obtain similar ES-like cells from neonatal mouse testis and these cells were similar to the ES/EG cells except that in their genomic imprinting pattern.

Spermatogenesis is a highly specialized process of cellular differentiation to produce spermatozoa. This differentiation process accompanies morphological changes that are controlled by a number of genes expressed in a stage-specific manner during spermatogenesis. Here we show that in mice, the absence of a testis-specific, cytoplasmic polyadenylate [poly(A)] polymerase, TPAP, results in the arrest of spermiogenesis. TPAP-deficient mice display impaired expression of haploid-specific genes that are required for the morphogenesis of germ cells. The TPAP deficiency also causes incomplete elongation of poly(A) tails of particular transcription factor messenger RNAs. Although the overall cellular level of the transcription factor TAF10 is unaffected, TAF10 is insufficiently transported into the nucleus of germ cells. We propose that TPAP governs germ cell morphogenesis by modulating specific transcription factors at posttranscriptional and posttranslational levels., Poly(A) tails of eukaryotic mRNAs are implicated in various aspects of mRNA metabolism, including transport into the cytoplasm, stability, and translational control (1, 2). Thus, the control of poly(A) tail [...]

Here we investigated the pathogenesis of deletion mutant mitochondrial (mt)DNA by generating mice with mutant mtDNA carrying a 4696-basepair deletion ([DELTA]mtDNA4696), and by using cytochrome c oxidase (COX) electron micrographs to identify COX activity at the individual mitochondrial level. All mitochondria in tissues with [DELTA]mtDNA4696 showed normal COX activity until [DELTA]mtDNA4696 accumulated predominantly; this prevented mice from expressing disease phenotypes. Moreover, we did not observe coexistence of COX-positive and -negative mitochondria within single cells. These results indicate the occurrence of inter-mitochondrial complementation through exchange of genetic contents between exogenously introduced mitochondria with [DELTA]mtDNA4696 and host mitochondria with normal mtDNA. This complementation shows a mitochondria-specific mechanism for avoiding expression of deletion-mutant mtDNA, and opens the possibility of a gene therapy in which mitochondria possessing full-length DNA are introduced., Author(s): Kazuto Nakada [1, 2, 3, 6]; Kimiko Inoue [1, 5, 6]; Tomoko Ono [1]; Kotoyo Isobe [1]; Atsuo Ogura [5]; Yu-Ichi Goto [4]; Ikuya Nonaka [3]; Jun-Ichi Hayashi (corresponding [...]